title: Using molecular beacons to study dispersal of mRNPs from the gene locus.
abstract: Before leaving the site of transcription, newborn messenger RNAs (mRNAs) become associated with a number of different proteins. How these large messenger ribonucleoprotein (mRNP) complexes then move through the dense nucleoplasm to reach the nuclear periphery has been a fascinating question for the last few years. We have studied the mechanism of this process by tracking individual mRNPs in real time. We were able to track mRNPs at single-molecule resolution because we utilized mRNAs that were engineered to have a sequence motif repeated 96 times in their untranslated region. These mRNAs were visualized with the help of molecular beacons that were specific for the repeated sequence; the binding of 96 molecular beacons to each mRNA molecule rendered them so intensely fluorescent that they were visible as fine fluorescent spots that could be tracked by high-speed video microscopy. In this chapter, we describe the details of the construction of genes containing the tandem repeats, the integration of such genes into the genome of a cell line, the design and testing of molecular beacons, time-lapse imaging of mRNPs, and computer-aided generation and analysis of the tracks of the individual mRNPs. These methods will be useful for studies of other dynamic processes such as mRNA export, splicing, and decay.
authors.affiliation: Public Health Research Institute, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
authors.forename: Patrick T C
authors.initials: PT
authors.lastname: van den Bogaard
authors.forename: Sanjay
authors.initials: S
authors.lastname: Tyagi
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein